b-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with ...

13 Pages·2008·1.12 MB·English

b-Subunits of the SnRK1 Complexes Share a Common
Ancestral Function Together with Expression and
Function Specificities; Physical Interaction with
Nitrate Reductase Specifically Occurs
via AKINb1-Subunit1[C][OA]
Ce´cile Polge2, Mathieu Jossier, Pierre Crozet, Lionel Gissot3, and Martine Thomas*
Laboratoire Signalisation et Re´gulation Coordonne´e du Me´tabolisme Carbone´ et Azote´, Institut de
Biotechnologie des Plantes (UMR8618), Universite´ Paris-Sud, F–91405 Orsay cedex, France
TheSNF1/AMPK/SnRK1kinasesareevolutionaryconservedkinasesinvolvedinyeast,mammals,andplantsinthecontrolof
energybalance.Theseheterotrimericenzymesarecomposedofonea-typecatalyticsubunitandtwog-andb-typeregulatory
subunits.Inyeastithasbeenproposedthattheb-typesubunitsregulateboththelocalizationofthekinasecomplexeswithin
the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three b-type subunits of
Arabidopsis(Arabidopsisthaliana;AKINb1,AKINb2,andAKINb3)restorethegrowthphenotypeoftheyeastsip1Dsip2Dgal83D
triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINb promoter::
b-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs,
developmental stages, and environmental conditions. Finally, our results show that the b-type subunits are involved in the
specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation
data,theystronglysupporttheproposalthatnitratereductaseisarealtargetofSnRK1inthephysiologicalcontext.Altogether
our data suggest the conservation of ancestral basic function(s) together with specialized functions for each b-type subunit
inplants.
TheSNF1/AMPKkinasefamilyplaysanimportant kinase, once activated by an increase in AMP level,
roleinthecontroloftheenergybalance.Inyeast,SNF1 switches off ATP-consuming pathways such as fatty
(forSucnonfermenting1)isasakeyplayerintheshift acid, cholesterol, and protein syntheses, and switches
fromfermentativetooxidativemetabolisminresponse onATP-producingpathwayssuchasfattyacidoxida-
to Glc deprivation (diauxic shift; Hardie et al., 1998). tion andglycolysis(Hardie,2004). Atthewhole-body
Indeed, a quarter of the genome presents a SNF1- level, AMPK is activated in response to muscle con-
dependent change in expression during this shift traction, resultingin an increased fatty acid oxidation
(Young et al., 2003). In mammals, AMPK, the SNF1 and Glc uptake (Hardie and Carling, 1997; Merrill
homolog,hasbeeninvolvedinsensingthecellularand et al., 1997; Fryer et al., 2002). This enzyme also plays
whole-body energy levels. At the cellular level, this an important role in Glc homeostasis since it partici-
pates in the inhibition of insulin production and
secretion by the islet b cells when Glc level in blood
1This work was supported by the Ministe`re de l’Education
is low (da Silva Xavier et al., 2000, 2003) and to the
NationaleetdelaRecherche,France(toC.P.,M.J.,L.G.,andP.C.).
inhibition of Glc metabolism in liver (Foretz et al.,
2Present address: Laboratoire de Bioe´nerge´tique Fondamentale
1998; Leclerc et al., 1998; for review, see Rutter et al.,
et Applique´e, INSERM U884, Universite´ Joseph Fourier, F–38041
2003). AMPK also regulates energy intake, its activa-
Grenoble,France.
3Presentaddress:LaboratoiredeBiologieCellulaire,Laboratoire tion in hypothalamus leading to a stimulation of
CommundeCytologie,INRAVersailles,RD10,RoutedeSaintCyr, food intake (Andersson et al., 2004; Minokoshi et al.,
F–78026Versaillescedex,France. 2004). In plants, several reports have involved SnRK1
*Correspondingauthor;[email protected]. (for SNF1-related protein kinase 1) in the regulation
Theauthorresponsiblefordistributionofmaterialsintegraltothe of metabolic pathways (for review, see Polge and
findings presented in this article in accordance with the policy Thomas, 2007). Indeed, Suc-P synthase,nitrate reduc-
describedintheInstructionsforAuthors(www.plantphysiol.org)is:
tase (NR), and TPS5 (for trehalose-P synthase 5) have
MartineThomas([email protected]).
been identified as in vitro phosphorylation targets of
[C] Somefiguresinthisarticlearedisplayedincoloronlinebutin
SnRK1 (Sugden et al., 1999; Harthill et al., 2006).
blackandwhiteintheprintedition.
[OA]Open Access articles can be viewed online without a SnRK1 kinases also regulate the expression of several
subscription. genes involved in carbohydrate metabolism such
www.plantphysiol.org/cgi/doi/10.1104/pp.108.123026 as Suc synthase and a-amylase (Purcell et al., 1998;
1570 Plant Physiology, November 2008, Vol. 148, pp. 1570–1582, www.plantphysiol.org (cid:1) 2008 American Society of Plant Biologists
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AKINb-Subunits: Functional Redundancy and Diversity
Laurieetal.,2003)andisinvolvedintheregulationof sion, a potato (Solanum tuberosum) b-type subunit,
starch synthesis (Zhang et al., 2001; Geigenberger, leadstoanabnormaldevelopmentofrootsandtubers
2003; Thelander et al., 2004). Recently a major contri- (Lovasetal.,2003),suggestinganimportantroleofthis
bution was brought by the work of Baena-Gonza´lez subunitinplantdevelopment.Inyeast,theb-subunits
and collaborators using transient expression experi- have been proposed to mediate the interaction of the
ments and transgenic plants. Their data implicate the kinase with its targets (Vincent and Carlson, 1999).
two Arabidopsis (Arabidopsis thaliana) SnRK1 kinases Moreover, the authors proposed that each b-subunit
ascentralintegratorsoftranscriptionalnetworksinre- wouldinteractwithspecificsetsoftargetsandthatthis
sponsetostressandenergysignaling(Baena-Gonza´lez specificity is dependent on their variable N-terminal
et al., 2007), thus reinforcing diverse previous reports region. b-Type subunits could also regulate the local-
(Nemethetal.,1998;Farrasetal.,2001;Bradfordetal., izationofthekinasecomplexeswithinthecell,through
2003; Buitink et al., 2003; Hao et al., 2003; Thelander theirN-terminalandGBDregions(Vincentetal.,2001;
etal., 2004;Gissot et al., 2006; Radchuket al., 2006). Wojtaszewski et al., 2002; Hedbacker et al., 2004). In
In all eukaryotic kingdoms, SNF1/AMPK/SnRK1 plants, it has been shown recently that Arabidopsis
kinases function as heterotrimeric complexes com- AKINb1 and AKINb2 are N-myristoylated and that
posed of one catalytic subunit, the a-type subunit, their subcellular localization may vary depending on
and two regulatory subunits, the g- and b-type sub- this mechanism together with changes in kinase ac-
units(Daviesetal.,1994;Mitchelhilletal.,1994;Jiang tivity (Pierre et al., 2007). These data suggest that
andCarlson,1997;Boulyetal.,1999).Inmammals,the b-subunits might play a major role in the regulation
formationoftheheterotrimerisnecessaryforAMPKa andspecializationoftheSNF1complexes.Forinstance,
activity(Dycketal.,1996;Woodsetal.,1996).Inyeast, SNF1complexeshavebeeninvolvedinvariousaspects
SNF4 (the g-subunit) deletion or simultaneous dele- oftheinvasivefilamentousyeastgrowthdependingon
tions of the three b-subunits totally inactivates the the b-subunit integrated in the complex (Vyas et al.,
SNF1activityinvivo(Carlsonetal.,1981;Schmidtand 2003).
McCartney,2000),indicating that theyallplayimpor- Yeast SNF1, mammalian AMPK, and plant SnRK1
tant roles within the complex. kinases are now considered as global metabolic regu-
Concerning the b-type subunits, the conservation lators. To gain more insights into the role of plant
among the yeast, mammal, and plant SIP/AMPKb/ SnRK1 complexes, we have focused our study on the
SnRKb proteins spread along three regions, the ASC three b-type subunits of Arabidopsis, AKINb1,
(forassociationwithSnf1complex),theKIS(forkinase- AKINb2, and AKINb3. We demonstrate that these
interacting sequence), and the GBD (for glycogen- three subunits restore the phenotype of the yeast
binding domain) domain. In yeast, the ASC domain, sip1Dsip2Dgal83Dmutant,suggestingtheconservation
locatedattheCterminus,allowstheinteractionofthe of an ancestral basic function(s). However, each of
b-subunits (SIP1, SIP2, GAL83) with SNF4 (Jiang and thesethreesubunitspresentsaspecificpatternofgene
Carlson, 1997). The internal KIS region allows the expression during development and in response to
interaction of the b-subunits with SNF1 (Jiang and different environmental conditions. Finally, we report
Carlson, 1997). More recently, another domain, over- the first evidence, to our knowledge, of a physical
lappingthepreviouslydefined yeastKISdomainand interactionbetweenthemajorformofNR,NR2,anda
presenting the characteristics of an N-isoamylase do- SnRK1 complex containing AKINb1. b-Specific ex-
main,hasbeendescribed(Hudsonetal.,2003).Sincein pressiondata,togetherwiththespecificinteractionof
some cases this region has been shown to bind to AKINb1withNR2,stronglysuggestspecializedfunc-
glycogen, it has been named GBD (Polekhina et al., tions foreach b-type subunitin plants.
2003).
Plantb-subunitscanbegroupedintwoclasses.One
class of two plant b-subunits is composed of proteins
presentingthecharacteristicsofthethreeyeastandthe RESULTS
two mammalian b-subunits in that they all have the
AKINb1, AKINb2, and AKINb3 Share a Common
three conserved domains previously described (Yang
Ancestral Function(s)
et al., 1992; Erickson and Johnston, 1993; Bouly et al.,
1999; Buitink et al., 2003; Kemp et al., 2003). In To get insights into the function of the plant
Arabidopsis, the two proteins of this class, AKINb1 b-subunits,we have performed yeast functionalcom-
and AKINb2, present an overall identity of 49% and plementation experiments using a yeast b-subunit
55% within these regions (Bouly et al., 1999). The triple mutant (Schmidt and McCartney, 2000). The
secondclassiscomposedofshorterb-subunits,named yeast strain presents complete deletions of the three
AKINb3 in Arabidopsis, lacking the GBD and the yeast b-subunits (sip1Dsip2Dgal83D) leading to a snf
N-terminal regionof the protein (Gissot et al., 2004). phenotype, and thus is unable to grow on alternative
Several studies have highlighted an emerging im- carbon sourcessuch asethanol and glycerol(Schmidt
portance of the b-type subunits within the complex, andMcCartney,2000).Wehavepreviouslyshownthat
making them particularly attractive. For instance, in the plant-specific subunit AKINb3 can complement
plants, a 90% to 95% decrease in StubGAL83 expres- this mutant (Gissot et al., 2004). In Figure 1, we show
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Polge et al.
that the two other Arabidopsis b-subunits, AKINb1 is present (Twell et al., 1991; Fig. 2). However, this
andAKINb2,canalsorestorethegrowthabilityofthe sequence differs slightly from those previously iden-
mutant on a medium containing glyceroland ethanol tified(Twelletal.,1991;Weteringsetal.,1995;Bateand
as carbon sources. This result suggests that plant Twell, 1998; TGTTGGTT instead of TGTGGTT). One
b-type subunits can substitute for any of the endoge- sugar-repressivebox(Moritaetal.,1998)hasalsobeen
nous subunit to form a chimeric, active enzymatic found inthis promoter (Fig. 2).
complex,andsharewithyeastsubunitsoneorseveral TheAKINb3promoter(976bpfromtranscriptionstart
common ancestral functions. However, the growth site)contains17pollenboxes(Fig.2).SevenAAATGA
restorationappearsonlypartialwithAKINb2despite boxes are on the major strand and three on the minus
its structural similarity with AKINb1 and with the strand (Weterings et al., 1995) as well as three LAT56-
yeast b-subunits. typepollenboxes(Twelletal.,1991).Adarkinduction
box(Deheshetal.,1990)ispresentintheexonicregion
locatedbetweenthetwoleaderintrons(Fig.2).
Analysis of AKINb1, AKINb2, and AKINb3 Genes
The detailed analysis of the promoters of the three
Reveals Different Potential Regulatory Elements
AKINbgenessuggestsadifferentialregulationofgene
To determine whether the three plant b-type sub- expression.Toconfirmthisassessmentinvivo,wehave
units could have redundant functions, as suggested studied the expression patterns of the three genes in
bytheyeastfunctionalcomplementationexperiments differentorgans,atdifferentdevelopmentalstages,and
describedabove,wehaveundertakenadetailedstudy inresponsetolightconditionsandtosugaravailability.
of their promoterregions(Fig. 2).
The AKINb1 promoter (772 bp from transcription AKINb1, AKINb2, and AKINb3 Present Specific Patterns
startsite)containsone6-bpconsensussequenceknown
of Expression during the Course of Plant Development
asauxinresponseelement(Ulmasovetal.,1997;Fig.2).
However, digital northern analysis carried out with Toinvestigateexpressionpatternsindifferentorgans
Genevestigator (Zimmermann et al., 2004; https:// and at different developmental stages, we have pro-
www.genevestigator.ethz.ch)didnotrevealanypoten- duced AKINb promoter::GUS transgenic lines. Trans-
tial regulation by auxin. One pollen box is present lationalfusionsweremadebetweentheUIDAreporter
(Twelletal.,1991),aswellastwodarkinductionorlight geneandfragmentsofAKINb1,AKINb2,andAKINb3
repressionboxes(Deheshetal.,1990;Inabaetal.,2000; promoterscorresponding,respectively,to772,998,and
Fig.2).Twoantagonistictypesofboxeshavealsobeen 976bpupstreamofthetranscriptionstart,andcontain-
foundinthispromoter,sixsugar-repressiveboxes(Lu ing the subset of regulation motifs in these regions
etal.,1998;Moritaetal.,1998)andonesugar-inducible describedinFigure2.Stainingdata,showninFigure3,
box(Sunetal.,2003;Fig.2). are representative of the results obtained for at least
IntheAKINb2promoter(998bpfromtranscription threeAKINb1::GUS,AKINb2::GUS,andAKINb3::GUS
startsite),adomainsimilartotheLAT56pollenmotif stablehomozygoustransgeniclines.
GUS staining experiments indicate that there is at
least one b-subunit expressed in all the organs and
tissues analyzed (Fig. 3D). Some organs and tissues
expressthethreeAKIN::GUSconstructs.Thisisfound
in the cotyledons soon after germination (data not
shown)andinrootsfrom72-h-oldseedlings(Fig.3A,
photographs b), in the primordia of roots and repro-
ductive organs (Fig. 3B), in the stamen filament and
thepistilofyoungbuds,andinmaturepollen(Fig.3B,
photographs f). These results suggest the existence of
threetypesofSnRK1complexesinthesetissues,ab1g,
ab2g,andab3g(arepresentinga1ora2andgbeingg
or bg). Interestingly there are also tissues where the
expressionofonlyone b-subunit isdetected. AKINb1
is mainly expressed in the sepals of young buds (Fig.
3B,photographs1cand1d)andintheovarywall(Fig.
3B, photographs 1e and 1f). AKINb2 is themainform
Figure1. Complementationoftheyeastsip1Dsip2Dgal83Dtriplemu- in stigmata, in placenta with a strong coloration in
tantbyAKINb1,AKINb2,andAKINb3.Theyeastsip1Dsip2Dgal83D septum(Fig.3B,photographse),infloralpedicel,and
mutantwastransformedwithArabidopsisAKINb1,AKINb2,orAKINb3
in the insertion area of the floral organs (Figs. 3B
coding sequence. Two serial dilutions were plated on YPD plates
[photographse]and4A).AKINb3isthemainformin
supplementedwithGlc2%(Glc)orglycerol-ethanol3%to2%(Gly/
ethanol)ascarbonsources.Thecellsweregrownat30(cid:3)Cfor4d.The theroottipof48-h-oldseedlings(Fig.3A,photograph
wild-type(WT)strainwasusedasacontrol.sip1Dsip2Dgal83Disthe 3a), in leaf primordia, in developing pollen, in ovules
untransformedtriplemutantstrain.[Seeonlinearticleforcolorversionof (Fig. 3C, photographs 3e and 3f), and in immature
thisfigure.] seeds (Fig. 3C,photograph 3g).
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AKINb-Subunits: Functional Redundancy and Diversity
Figure2. PositionsoftheregulationboxesidentifiedinthepromotersofAKINbgenes.Thefigureshowsthepositionsofthe
regulatorysequencesidentifiedamongthosedescribedintheliterature.Allpositionsarereferencedtothetranscriptionstartsite
(t.s.).Exonsareboxed,hatchedboxesrepresent5#UTR,andbarsindicatepromoterregionsexceptforthoselabeledI andI
1 2,
representingleaderintrons.Bracesshowthesequencesusedforthepromoter::GUSconstructs.Pollen-specificsequencesare
represented by squares; full squares are for LAT56 (TGTGGTT; Twell et al., 1991) and gray squares are for AAATGA box
(AAATGA;Weteringsetal.,1995).Darkinductionorlightrepressionboxesarerepresentedbytriangles;fulltrianglesareforDE1
dark-inducibleelement(phytochromedependent;GGATTTTACAGT;Inabaetal.,2000)andgraytrianglesareforGT2sequence
(GGTAATT;Deheshetal., 1990).Sugarrepressionsequencesarerepresentedbycircles;fullcirclesareforsugar-repressive
element(TTATCC; Lu et al., 1998),graycirclesare for a-amylase (TATCCAT; Moritaet al., 1998),andwhitecirclesare for
pyrimidinebox(CCTTTT;Moritaetal.,1998).SugarinductionSUSIBA2bindingmotifisinboldcircles(TGACT;Sunetal.,
2003). Auxin response elements are represented by diamond shapes (TGTCTC; Ulmasov et al., 1997). The Arabidopsis
Information Resource accession numbers for the genes encoding for AKINb1, AKINb2, and AKINb3, respectively, are
At5g21170,At4g16360,andAt2g28060.
Altogether,thestainingdatashowedthatatleastone cence to allow the abscission of floral parts. The
b-subunitisexpressedineachorganortissuetested(Fig. increase of AKINb2::GUS expression could be related
3D), suggesting a ubiquitous presence of the AKINb- to a program of cell death. The expression of AKIN
typeproteins in thewholeplant.Thus, thesedataalso genes during the process of senescence was thus
indirectly suggest the presence of AKIN kinase com- followed by northern-blot experiments at different
plexes in all the organs of Arabidopsis since we have leaf developmental stages (named a–i), throughout
previouslyshownthatboththecatalyticAKINa-subunit the development of the plant (Fig. 4, B and C). Our
and AKINbg, one AKINg-subunit, appear constitu- results show that expression of all three b-type sub-
tivelyexpressed(Gissotetal.,2006).Anotherinteresting units is up-regulated at the advanced stages of senes-
resultwasthefactthatinsomeorgansthethreeb-type cence when most RNAs are degraded, as shown by
subunits present specific patterns of expression that hybridization with EF1a and by the amount of intact
moreover vary during organ development. It is note- 28SRNA(Fig.4C,stagesg,h,andi).Interestingly,our
worthy that, in opened flowers and siliques, there is results also show that the mRNA levels of the other
nearlynooverlapbetweenthepatternsofexpressionof membersoftheAKINcomplex,AKINa1andAKINbg,
thethreeb-typesubunits(Fig.3C). aremaintainedatthesestages,stronglysuggestingthe
presence of different functional AKIN kinase com-
AKINb1, AKINb2, and AKINb3 Are Up-Regulated plexesduringsenescenceandthuspotentiallyseveral
during Senescence roles ofthe kinaseinthis process.
During the analysis of AKINb2-GUS expression AKINb Genes Are Differently Regulated by Light
pattern, a strong staining was detected in the pedicle
duringflowerdevelopment(Fig.4A).Itiswellknown Regulatory sequences associated with dark induc-
that in this region, after flowering, cells enter senes- tion have been identified in AKINb1 and AKINb3
Plant Physiol. Vol. 148, 2008 1573
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Copyright © 2008 American Society of Plant Biologists. All rights reserved.
Polge et al.
Figure 3. Histochemical localization of
GUS activity in transgenic Arabidopsis
plants containing AKINb-GUS fusions in
differentorgansandduringdevelopment.
A, Seedling development of the AKINb-
GUSArabidopsistransformants.a,Germi-
nation;b,wholeseedling;b’,zoomonthe
shootapicalmeristemarea.B,Flowerbud
development. c, Inflorescence; d and e,
buds artificially opened for the photo-
graphs;f,openedflowers;theinsetsshow
GUSstaininginthepollen.C(g),Flowers
and siliques, cleared by chloralhydrate
treatment. D, Summary of GUS staining
data observed in different organs and
duringdevelopment ofArabidopsis.Each
color represents the expression of one,
two, or the three AKINb genes, blue for
AKINb1, yellow for AKINb2, red for
AKINb3,greenforAKINb1andAKINb2,
orangeforAKINb2andAKINb3,anddark
blue for AKINb1, AKINb2, and AKINb3.
1(a–g),AKINb1;2(a–g),AKINb2;3(a–g),
AKINb3.
promoters, reinforcing, for AKINb1, previous data determine whether the observed variations in mRNA
fromour laboratoryshowingdifferentiallight regula- levels are due to a transcriptional regulation, as
tion(Boulyetal.,1999).Indeed,arapidincreaseinthe suggested for AKINb1 and AKINb3 by the presence
AKINb1 transcript level in the dark had been de- ofdarkinductionboxes,experimentswereperformed
scribed, an effect that is reversed by light. No infor- usingthe AKINb::GUS transgeniclines described ear-
mationisavailableconcerningAKINb3regulation.To lier. GUS activities were measured in 3-week-old
1574 Plant Physiol. Vol. 148, 2008
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AKINb-Subunits: Functional Redundancy and Diversity
Figure 4. Levels of AKINb transcripts during
senescence.A,ExpressionpatternofAKINb2::
GUS in the pedicel. Histochemical localiza-
tionofGUSactivity.B,Positionanddevelop-
mentalstagesoftheleavesusedfornorthern
analyses.Arabidopsisseeds,ecotypeColum-
bia(C0),weregrowninsoilat21(cid:3)Cunder16-
h-light/8-h-darkcycle(longday).Leaveswere
taken from different developmental stages. a
andb,Leavesof,respectively,8/10-leafor13/
14-leafrosettes.ctoe,1-,2-,and4-cm-long
leaves, respectively, from a 10-cm-diameter
rosette. f and g, Leaves from a plant at the
beginning of the flowering period. h, Oldest
rosette leaves from a flowering plant; and i,
oldest leaves at the end of the flowering
period. C, Northern blots experiments. Full-
lengthAKINandEF1awereusedtoprobethe
RNAblots.28SRNAarevisualizedunderUV
light illumination of the agarose gel stained
withethidiumbromide.rRNAandexpression
levelofEF1aattesttothelevelofdegradation
oftheRNAandthusthesenescentcondition
oftheleavesanalyzed.
plantlets after they were held for 2.5 d in the dark or the dark in two transformants (Fig. 4C). This result,
kept for 2.5 d under a 16-h photoperiod. Plants were together with the presence of a dark box in its pro-
harvested in the middle of the 16-h light period. For moter, suggests that AKINb3 is transcriptionally reg-
each construct, three independent transgenic lines ulated in these conditions. However, this dark box is
were used, and for each transgenic plant, two inde- differentfromtheonepresentintheAKINb1promoter
pendent experiments were performed with measure- that was described as phytochrome dependent, sug-
ments made in triplicate (Fig. 5). The results show an gesting that these two promoters could be dark regu-
increaseofGUSexpressionofaround1.5to2whenthe latedbydifferentmechanisms.Sincenovariationwas
plantlets are kept in the dark for 2.5 d, indicating a observed for AKINb2, the observed differential ex-
transcriptional regulation of AKINb1 in these condi- pressioneffectisverylikelylinkedtotheAKINb1and
tions(Fig.5A).Theyarereinforcedbyexpressiondata AKINb3 promoters.
on Genevestigator. In experiments 109 and 56 (exper-
iment 109, AtGenExpress response to different light AKINb1 Expression Is Regulated by Sugar and
treatment; and experiment 56, gene expression and
Light Independently
carbohydrate metabolism through the diurnal cycle;
Smith et al., 2004), the AKINb1 transcript level in- Thedark-inducedvariationsofexpressionobserved
creases 1.3- and 3.3-fold after 45 and 60 min in the can be a direct consequence of dark/light conditions,
dark,respectively. as suggested by the presence of a dark-responsive
NosignificantvariationofAKINb2::GUSexpression element (phytochrome dependent) in the AKINb1
inresponsetodarknesswasdetectedusingtheexper- promoter (Inaba et al., 2000). Nevertheless, since the
imental conditions described (Fig. 5B). promoter also contains sugar-responsive elements, it
ForAKINb3::GUS,GUSactivityshowsanaverageof couldalsobeanindirectconsequenceofthevariation
1.6-to2.2-foldincreaseingeneexpressionafter2.5din of sugar content occurring during the dark/light pe-
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Polge et al.
Figure 5. Dark/light regulation of AKINb gene ex-
pression.A,B,andC,GUSactivitiesoftheAKINb1::
GUS, AKINb2::GUS, and AKINb3::GUS transform-
ants, respectively, in themiddleofthe photoperiod
(light)orafter2.5dofdark.Invitro-culturedplants
were grown on half-strength Murashige and Skoog
mediasupplementedwith20g/LSucat20(cid:3)Cundera
16-h-light/8-h-darkregimefor3weeks.Plantswere
then maintained under this regime for 2.5 d or
transferredtodarkfor2.5d(darkness)andcollected
inthemiddleofthelightperiod(light).Datarepresent
themean6SDoftwoindependentexperimentsand
triplicate measurements. Asterisks indicate signifi-
cantdifferencesbetweenexpressionindarkandlight
conditions(**,,0.01and*,,0.05,Student’sttest).
riod. Thus, the effects of dark and sugar on AKINb1 The same experiment has been performed with
gene expression were tested. Three-week-old plant- AKINb3::GUS transgenic lines, but no significant var-
lets,grownonone-halfMurashigeandSkoogmedium iation was measured in the presence of Suc in our
supplemented with 5, 20, or 40 g/L Suc, were sub- conditions (data not shown) nor reported in the
jectedornotto2.5dinthedark.Inthelight,theGUS Genevestigator database. These results suggest that,
activity, measured in AKINb1::GUS transgenic lines, incontrasttoAKINb1,AKINb3geneexpressionisonly
decreased as a consequence of the increase in the regulated by a dark signal and is sugar independent.
amountofSucinthemedium(Fig.6A).TheAKINb1-2, Altogether, these data suggest that expression of the
AKINb1-4,andAKINb1-5lines showed,respectively, AKINb genes is related to the presence/absence of
4.9-, 2.1-, and 7.3-fold decreases in GUS activity dark/sugar regulatory domains in their promoter
when plantlets were grown on one-half-strength sequences.
MurashigeandSkoog40comparedtoone-half-strength
Murashige and Skoog 5. These data suggest the exis-
MembersoftheAKINKinaseComplexInteractwithNR
tenceofasugardose-dependentinhibitoryresponseof
AKINb1 gene expression. They are strengthened by Since the b-type subunits of Arabidopsis present
expressiondatafromexperiment15ofGenevestigator, differential gene expression, we have hypothesized
which indicated a 2.5-fold decrease of AKINb1 ex- that they could interact specifically with different
pression when seedlings were grown with Suc in targets, as previously proposed in yeast (Vincent and
the medium compared to medium without Suc (M. Carlson, 1999). We have tested this hypothesis by
Campbell, NASCArrays; Craigon et al., 2004). How- analyzing the interaction between the AKIN subunits
ever, when the same experiment was performed after and NR2, which is phosphorylated in vitro by SnRK1
2.5 d in the dark, the repressive effect of sugar (Sugden et al., 1999). With this aim, the full-length
remained but was more variable. So, while the trend cDNAs of the AKIN subunits and NR2 genes were
isclear, itis statistically notsignificant when compar- clonedinpGBKT7andpGADT7vectorstoperformin
ingone-half-strengthMurashigeandSkoog5andone- vitrointeractionassays(Fig.7A)andtwo-hybrid(Fig.
half-strength Murashige and Skoog 40, except for the 7C)experiments.Fortheinvitrointeractionassays,we
AKINb1-2line(Fig.6B).Intheseconditionssugardoes took advantage of the fact that NR2 directly binds to
not appear to be sufficient to balance the inductive ProteinA-Sepharose (without any antibody added). It
effect of a dark period on AKINb1 gene expression. was found that the NR2-linked ProteinA-Sepharose
This suggests that sugar inhibition occurs only in binds AKINa2, AKINb1, and, to a lesser degree,
normal photoperiod growth conditions and that the AKINb3, while protein A alone does not show any
inducible effect of dark and the repressive effects of retention (Fig. 7A). Two-hybrid experiments con-
sugar can be uncoupled. Altogether, these data sug- firmed the existence of a strong interaction between
gestthatAKINb1geneexpressionissubjectedtoboth NR2andAKINb1andofaweakerinteractionbetween
light and sugar signals independently. NR2 and AKINb3 (Fig. 7D). A significant interaction
1576 Plant Physiol. Vol. 148, 2008
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Copyright © 2008 American Society of Plant Biologists. All rights reserved.
AKINb-Subunits: Functional Redundancy and Diversity
of the protein. Such a mechanism was also proposed
by Vincent and Carlson (1999) in yeast since the
deletion of the N-terminal part on SIP2 allows the
interaction of the remaining fragment with SIP4, a
partnerofGAL83. Totestthishypothesis,two-hybrid
and in vitro interaction experiments have been per-
formed using NR2 and different subdomains of the
threeb-typesubunits(Fig.7,B–D).Ourdatashowthat:
(1)Full-sizeAKINb1stronglyinteractswithNR2while
full-size AKINb2 does not; (2) when deleted from its
N-terminalregion,AKINb1KIS/ASC(C-terminalpart
oftheprotein)stillinteractsbuttoamuchlesserdegree,
andAKINb2KIS/ASCnowshowsaninteractionwith
similarstrength(Fig.7,BandD);and(3)theN-terminal
domainsofneitherAKINb1norAKINb2interactwith
NR2 (Fig. 7B). Thus, AKINb1 and AKINb2 bind NR2
through their C-terminal domains (KIS-ASC). In the
case of full-length AKINb1, the N-terminal domain
confers a greater binding affinity for NR to the holo-
protein.TheAKINb2N-terminaldomaincouldhavean
opposite effect since no interaction is observed with
NR2 (Fig. 7D). This could be due to conformational
effectsthatinfluencethebindingprocesspositivelyand
negatively,fortherespectiveproteins.
Figure 6. Regulation of AKINb1 gene expressionbydark and sugar.
Effects of Suc on GUS expression from AKINb1::GUS transformants DISCUSSION
duringthephotoperiod(A)andafter2.4dinthedark(B).Arabidopsis
seeds were grown, in vitro, on half-strength Murashige and Skoog ThisarticlefocusesonthestudyoftheSnRK1b-type
mediadilutedtwicesupplementedwith5,20,or40g/LSucat20(cid:3)C subunitsinArabidopsistoattainabetterunderstand-
undera16-h-light/8-h-darkregimeduring3weeks.Plantswerethen ing of the involvement of these subunits in the func-
either transferred in the dark for 2.5 d (darkness) or kept under this tion(s)oftheplantSNF1kinases.Thisendeavorrelies
regimefor2.5dandcollectedinthemiddleofthelightperiod(light).
on the hypothesis proposed by Vincent and Carlson
Data represent the mean 6 SD of two independent experiments and that the b-type subunits mediate the interaction be-
triplicate measurements.Asterisksindicatesignificantdifferencesbe-
tween the kinase and its targets, imparting specific
tweenexpressionof plants grownon 20 or 40 g/L Suc compared to
plantsgrownon5g/LSuc(*,,0.05,Student’sttest). functions to the SNF1 kinase (Vincent and Carlson,
1999).
First, using GUS histochemical staining, we have
withAKINb2wasdetectedneitherbyinvitrobinding shown that in all Arabidopsis organs tested, at least
nor by two-hybrid assays (Fig. 7, A and D). The data oneb-typesubunitisexpressed.Thesedatasuggesta
indicatingthatNR2interactswiththekinaseAKINa2 ubiquitous presence of AKINb proteins and, thus,
(Fig. 7A, lane 2; and AKINa1, data not shown) rein- presumably of the AKIN complex in the whole plant
force the idea that NR2 is a real target of the SnRK1 since AKINa and AKINbg proteins appear constitu-
kinases.Moreover,theexistenceofastronginteraction tivelyexpressedinArabidopsis(Gissotetal.,2006;this
betweenNR2andAKINb1supportstheideathat,like work). Staining data also revealed that each b-type
inyeast,thekinasephosphorylatesitstargetsthrough subunit presents a specific, spatial pattern of gene
an interaction with the b-subunits. Furthermore, we expression in the different organs and temporally
show that, in the case of NR2, this mediation is during plant development. In some organs, the ex-
dependenton the b-subunit involved inthe complex. pressionofthethreeAKINbgenesoverlaps.Thisisthe
caseincotyledonsrightaftergermination,inleaves,in
roots, and in mature pollen. On the contrary, in some
In Plants, the N-Terminal Variable Region of the b-Type
organs, only one b-subunit is detected. It is particu-
Subunits May Confer the Specificity of Interaction with
larly striking in siliques where no overlap has been
the Targets of the Kinase
observedbetweentheexpressionpatternsofthethree
Taking into account that sequence homology be- AKINb genes.
tween AKINb1 and AKINb2 is mainly located in the We also show here that the dark/light-dependent
C-terminal region of these proteins (GBD, KIS, and variationsinAKINb1(Boulyetal., 1999) andAKINb3
ASC domains), and since AKINb2 was not able to transcripts occur at the transcriptional level, in good
interactwithNR2,wesuggestedthatthespecificityof agreementwiththepresenceofamotifassociatedwith
thisinteractionisdependentontheN-terminalregion darkinductioninthepromotersofthecorresponding
Plant Physiol. Vol. 148, 2008 1577
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Copyright © 2008 American Society of Plant Biologists. All rights reserved.
Polge et al.
Figure 7. Interaction of NR with members of the
AKINkinasecomplex.InteractionbetweenNR2and
membersoftheAKINkinasecomplexwasanalyzed
using in vitro cell-free interaction (A–C) and two-
hybrid (D) approaches. For the in vitro cell-free
interaction assay, NR2 was bound to ProteinA-
Sepharose.Interactionwithapartnerwasconsidered
positivewhenthebindingoftheputativepartnerwas
higher on the NR2-ProteinA-Sepharose than on the
ProteinA-Sepharosealone.Eachassayhasbeenper-
formedatleastthreetimes.OntheNR2lane,the+
and2indicate,respectively,presenceandabsence
ofNR2ontheProteinA-Sepharose;+coldindicates
that NR2 was not radiolabeled; since NR2 in vitro
translation products present an extra protein of the
same size as AKINa2 (approximately 58 kD), only
AKINa2 has been radiolabeled in the assay NR2
versus AKINa2. f, Control lane containing NR2
alone. A star indicates the position of the AKIN
subunit or subdomain tested. For each interaction
tested,resultsaresummarizedatthebottomoftables
(+forapositiveinteractionand2fornointeraction).
Since AKINb3 is constituted only by a KIS and an
ASCdomain,full-length(FL)andthe[KIS/ASC]frag-
mentcorrespondtothesamesequencesandthusthe
assay versus Nter (N terminus) does not exist. C,
Controlforcell-freeinteractionassaysusinglucifer-
ase,aproteinwhichdoesnotinteractwithNR2.On
theright,analiquotofluciferaseinvitrotranslation
assay (Tra) is used as a control of translation effi-
ciency. D, Two-hybrid results are representative of
eightclonesobtainedfromtwoindependentexper-
iments.ClonesareplatedonSD2LTH+3AT5mM,
at 30(cid:3)C. The interaction between AKINb3 and
AKINbg is a positive growth control. Due to the
autoactivation of BD-b2KIS and AD-b2KIS con-
structs,theinteractionbetweenNR2andb2KIShas
not been studied by this approach (ND, not deter-
mined).Thefirstpartofthesectioncorrespondstothe
interactionbetweenthedifferentsubdomainsofthe
AKINb-subunits(FL,fulllength,KIS/ASC,KIS,ASC)
fusedtotheADofGAL4andNR2fusedtotheBDof
GAL4. Since AKINb3[KIS/ASC] corresponds to
AKINb3FL, yeast clones have been plated only
once(=FL,sameas fulllength).Thesecondpartof
thesectioncorrespondstotheautoactivationcontrols
of these different subdomains against the empty
pGBKT7vector.
genes. Moreover, our results indicate that sugar in- sugar-repressive motifs). In contrast, no clear regula-
hibitsthistranscriptionalprocessbutsignificantlyless tion by sugar has been detected for AKINb3 in our
in the dark than in light. Our data suggest that it is conditions,whichisconsistentwiththeabsenceofany
possible to uncouple the effect of darkness and sugar known sugar-responsive elements in its promoter.
effectsonAKINb1geneexpression.Thus,thesesignals Altogether, the results of expression studies suggest
can act independently on AKINb1 expression as sug- that each b-type subunit may have specific functions.
gestedbythepresenceinAKINb1promoterofthetwo Such specific functions could have been conserved in
kinds of regulatory elements (dark-inductive and the plant kingdom since sugar regulation of AKINb1
1578 Plant Physiol. Vol. 148, 2008
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Copyright © 2008 American Society of Plant Biologists. All rights reserved.
AKINb-Subunits: Functional Redundancy and Diversity
expression (but not of AKINb2 and AKINb3) had getherwithadifferentialregulationofgeneexpression
previously been reported in germinating radicles of ledustothehypothesisthateachb-subunitcouldalso
Medicago truncatula. Indeed, up-regulation of AKINb1 havespecific functionsinrelationwith target binding
expressionuponstarvationwasrepressedinthepres- and correct positioning for phosphorylation by the
ence of Glc (Buitink et al., 2003). Furthermore, these catalytic subunit of the complex. We thus examined
datacanbelinkedtoresultsonthemossPhyscomitrella the interaction between the AKINb proteins and NR
patens SnRK1 kinases in the adaptation to light/dark (NR2;Sugdenetal.,1999).Cell-freeinteractionassays
transition, suggesting an activation of SnRK1 by Glc allowed us to detect an interaction between NR2 and
starvation (the dark period; Thelander et al., 2004). the two catalytic subunits of the AKIN kinase com-
More recently, AKINa1/2 proteins have been shown plex,AKINa1andAKINa2.Toourknowledge,thisis
tocontrolthereprogrammingofabroadarrayofgenes the first report of a physical interaction between a
in response to darkness and sugar conditions (Baena- SnRK1 kinase and NR, reinforcing the idea that this
Gonza´lez et al., 2007). Functional studies provide enzymeisatargetofSnRK1inplanta.Moreover,these
evidence of an inactivation of SnRK1 kinases in re- experiments reveal that NR2 also interacts with
sponsetoSuc,contrarytopreviousstudiessuggesting AKINb-subunits, this interaction being restricted to
an activation (Baena-Gonza´lez et al., 2007). Our data AKINb1 and to a much lower extent AKINb3. These
suggest that in Arabidopsis, a complex (or several data suggest that each b-type subunit could bind
complexes) containing AKINb1 could be involved in different panels of targets, thereby conferring to the
sucha function, in thedark. kinase complex specific physiological functions. In
We then wondered whether the Arabidopsis and the case of NR2, the interaction would occur via the
yeast b-subunits have identical functions. In yeast, the AKINb1-subunitwhoseexpressionisup-regulatedin
three b-subunitshavesomeredundant functions since response to darkness. It has been previously shown
only the complete deletion of the three b-subunits that thephosphorylationstate of NR2ishigher inthe
(sip1Dsip2Dgal83D mutant) leads to a snf phenotype, dark than in the light, this process being required for
unabletogrowonalternativecarbonsources(Schmidt its regulation. In darkened leaves NR is partially
andMcCartney,2000).Here,wereporttheabilityofeach inactivated following phosphorylation of the Ser-534
AKINb-subunit to restore the snf phenotype of the residueandbindingto14-3-3proteinsinthepresence
sip1Dsip2Dgal83D triple mutant, suggesting that the of cations (Weiner and Kaiser, 1999). Our results,
plantb-subunitshavesome(one)redundantfunctions together with cell-free phosphorylation data, support
with the yeast subunits. This also suggests that plant the idea that the NR phosphorylation in the dark
b-subunitsshareatleastoneconservedbasalfunction. occurs through one of the AKIN kinase complexes
This ancestral function could concern the control of containing the dark-induced subunit AKINb1, and
carbohydrate and energy metabolism. Indeed, in re- thusthatthiscomplexcouldbeamajoreffectorofNR
sponse to Glc or energy deprivation in yeast, SNF1 is down-regulation during the night.
necessaryforthetransitionfromfermentativetooxida- AKINb1 and AKINb2 present a high degree of
tivemetabolism(Hardieetal.,1998;Youngetal.,2003) identity, 49% in the whole protein, mainly located in
and AMPK in mammals plays a central role in the the conserved C-terminal KIS and ASC domains
energybalanceatthewholebodylevelbysynchronizing (55%). Is the N-terminal region, the most divergent
severalmetabolicpathways(Rutteretal.,2003;Carling, region, responsible for the differential interaction
2004). In plants, a recent work reports an inhibition of between AKINb1 and AKINb2 with NR2? To test
SnRK1 by sugars and strongly supports the idea that this hypothesis, we have performed two-hybrid and
SnRK1isacentralregulatorofplantmetabolismanden- cell-free binding experiments between NR2 and the
ergybalanceinresponsetosugarlevel(Baena-Gonza´lez different subdomains of each b-subunit. These exper-
etal.,2007).However,theregulationofSnRK1islikely imentsallowedustoshowthat,whendeletedfromits
morecomplexanddependentoftheorgananalyzed.For N-terminalregion,AKINb2caninteractwithNR2and
instance the situation appears different in crop sink that this interaction is mediated by the KIS and the
organs producing lots of starch compared to leaves. ASC domains. Indeed, together or alone, these do-
Indeed, in tubers, SnRK1 has been shown to regulate mains of AKINb2 can interactwith NR2, while in the
Suc-inducible events such as the Suc synthase gene presence of the N-terminal region no interaction was
expression(Purcelletal.,1998)andtheredoxactivation detected. Altogether, these data suggest that in the
oftheADP-Glcpyrophosphorylase,thekeyenzymeof case of NR2, the N-terminal domain of the AKINb1-
starchbiosynthesispathway(Geigenberger,2003).Nev- subunit:(1)playsanimportantpartinthespecificityof
ertheless,allthereportsagreewithanimportantroleof interaction with the target; and (2) is not involved
SnRK1 complexes in the regulation of carbohydrate directly in the interaction itself. We can hypothesize
metabolismandresourcepartitioning(Thelanderetal., that this variable region could allow the interaction
2004; McKibbin et al., 2006; Schwachtje et al., 2006; only with specific targets and/or reinforce this inter-
Baena-Gonza´lez et al., 2007; for review, see Polge and action, thus being responsible for the functional con-
Thomas,2007),likeinyeastandmammals. sequences of specific SnRK1 complexes according to
Specific patterns of expression obtained for each the b-subunit involved. In this case what could be
AKINb-subunit using GUS histochemical staining to- the function of AKINb3, the shorter plant-specific
Plant Physiol. Vol. 148, 2008 1579
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